The invention relates to regulation of the immune system.
The chemokines are a family of 8-12 kD proteins that regulate leukocyte trafficking by binding to specific seven transmembrane spanning G-protein-linked receptors. They can be divided into three families depending upon the sequence of conserved cysteine residues and this structural distinction corresponds to specific biologic properties in that the C-X-C, C-C, and C families are mainly chemoattractive for neutrophils, monocytes, and lymphocytes, respectively. Additionally, each chemokine family maps to a different chromosomal locus.
Eosinophils are circulating leukocytes that survive for several weeks. They dwell predominantly in tissues where they mediate pro-inflammatory and cytotoxic damage in selected diseases (e.g. asthma, parasitic infections, and malignancy). Given their presumed role in the pathogenesis of inflammatory states, the regulation of tissue recruitment of eosinophils is of interest and various chemoattractants have been found to be active on eosinophils, including leukotriene B4, platelet activating factor (PAF), and several chemokines (Resnick, et al. (1993) Amer. J. Resp. Cell. Mol. Biol. 8, 349-355). Chemokines active on eosinopbils include certain C-C chemokines: monocyte chemoattractive protein (MCP)-2 and 3, RANTES, and macrophage inflammatory protein (MIP)-1a (Rot, et al. (1992) J. Exp. Med 176, 1489-1495; Alam, R. et al. (1993) J. Immun. 150, 3442-3448; Dahinden, et al. (1994) J. Exp. Med. 179, 751-756; Weber, et al. (1995) J. Immun. 154, 4166-4172). A C-X-C chemokine, interleukin-8 (IL-8), is also chemoattractive for cytokine-primed eosinophils (Warringa et al. (1993) J. All. Clin. Immun. 91, 1198-1205). Notwithstanding their activity, none of these chemoattractive molecules are eosinophil specific and their relative importance in selected diseases and in experimental animal models of allergy remains unclear.
In general, the invention features substantially pure nucleic acid (for example, genomic DNA, cDNA, or synthetic DNA, or mRNA) encoding an eotaxin polypeptide as defined below. In related aspects, the invention also features a vector, a cell (e.g., a bacterial, yeast, nematode, or mammalian cell), a transgenic animal which includes such a substantially pure DNA encoding an eotaxin polypeptide of the invention or a knockout mutation in the eotaxin gene, and methods for modulating eosinophil chemotaxis.
In preferred embodiments, the eotaxin gene is the human eotaxin gene provided in FIG. 12, the murine eotaxin gene provided in FIG. 3A or the guinea pig eotaxin gene provided in FIG. 7. In various preferred embodiments, the cell is a transformed animal cell such as a human cell or a rodent cell.
In related aspects, the invention features a transgenic animal containing a transgene which encodes an eotaxin polypeptide and use of the eotaxin nucleotide sequence to engineer a transgenic animal having a knockout mutation in the eotaxin gene. The invention also features a cell that expresses the eotaxin gene. Preferably, the cell is an animal cell which is an epithelial or endothelial cell.
In a second aspect, the invention features a substantially pure DNA which includes a promoter capable of expressing the eotaxin gene in a cell. In preferred embodiments, the promoter is the promoter native to an eotaxin gene. Additionally, transcriptional and translational regulatory regions are preferably native to an eotaxin gene. A constitutive promotor or an inducible promotor are also included in the invention.
In other aspects, the invention features a substantially pure oligonucleotide including one or a combination of the sequences shown in FIGS. 3A, 7 and 12.
In a another aspect, the invention features a method of isolating an eotaxin gene or fragment thereof from a cell, involving: (a) providing a sample of cellular DNA; (b) providing a pair of oligonucleotides having sequence homology to a conserved region of an eotaxin gene (for example, oligonucleotides which include fragments of the sequences shown in FIGS. 3, 7, and 12 which are conserved as evidenced by the homologies shown in FIG. 3B); (c) combining the pair of oligonucleotides with the cellular DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and (d) isolating the amplified eotaxin gene or fragment thereof. Where a fragment is obtained by PCR, standard library screening techniques may be used to obtain the complete coding sequence.
In preferred embodiments, amplification is carried out using a reverse-transcription polymerase chain reaction, for example, the RACE method.
In another aspect, the invention features a method of identifying a eotaxin gene in a cell, involving: (a) providing a preparation of cellular DNA (for example, from the human genome); (b) providing a detectably-labelled DNA sequence (for example, prepared by the methods of the invention) having homology to a conserved region of an eotaxin gene; (c) contacting the preparation of cellular DNA with the detectably-labelled DNA sequence under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (d) identifying an eotaxin gene by its association with the detectable label.
In another aspect, the invention features a method of isolating an eotaxin gene from a recombinant DNA library, involving: (a) providing a recombinant DNA library; (b) contacting the recombinant DNA library with a detectably-labelled gene fragment produced according to the PCR method of the invention under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (c) isolating an eotaxin gene by its association with the detectable label.
In another aspect, the invention features a method of isolating an eotaxin gene from a recombinant DNA library, involving: (a) providing a recombinant DNA library; (b) contacting the recombinant DNA library with a detectably-labelled Eotaxin oligonucleotide of the invention under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (c) isolating an eotaxin gene by its association with the detectable label.
In another aspect, the invention features an eotaxin gene isolated according to the method involving: (a) providing a sample of cellular DNA; (b) providing a pair of oligonucleotides having sequence homology to a conserved region of an eotaxin gene; (c) combining the pair of oligonucleotides with the cellular DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and (d) isolating the amplified eotaxin gene or fragment thereof.
In another aspect, the invention features an eotaxin gene isolated according to the method involving: (a) providing a preparation of cellular DNA; (b) providing a detectably-labelled DNA sequence having homology to a conserved region of an eotaxin gene; (c) contacting the preparation of DNA with the detectably-labelled DNA sequence under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (d) identifying an eotaxin gene by its association with the detectable label.
In another aspect, the invention features an eotaxin gene isolated according to the method involving: (a) providing a recombinant DNA library; (b) contacting the recombinant DNA library with a detectably-labelled eotaxin gene fragment produced according to the method of the invention under hybridization conditions providing detection of genes having 50% or greater sequence identity; and (c) isolating an eotaxin gene by its association with the detectable label.
In another aspect, the invention features a method of identifying an eotaxin gene involving: (a) providing a mammalian cell sample; (b) introducing by transformation (e.g. viral, chemical, or mechanical transformation) into the cell sample a candidate eotaxin gene; (c) expressing the candidate eotaxin gene within the cell sample or isolating eotaxin from the tissue sample or protein isolated therefrom; and (d) determining whether the cell sample elicits an alteration in eosinophil chemotaxis, whereby an increased eosinophil specific chemotactic increase identifies an eotaxin gene.
In another aspect, the invention features an eotaxin gene isolated according to the method involving: (a) providing a cell sample; (b) introducing by transformation into the cell sample a candidate eotaxin gene; (c) expressing the candidate eotaxin gene within the tissue sample; and (d) determining whether the tissue sample elicits a eotaxin mediated response or decrease thereof, whereby a response identifies an eotaxin gene.
In another aspect, the invention features a method of detecting a eotaxin gene in a cell involving: (a) contacting the eotaxin gene or a portion thereof greater than 9 nucleic acids, preferably greater than 18 nucleic acids in length with a preparation of genomic DNA from the cell under hybridization conditions providing detection of DNA sequences having about 50% or greater sequence identity to the conserved DNA sequences of FIG. 3A, FIG. 7, or FIG. 12, or the sequences which are conserved among eotaxins relative to other proteins, as deduced from the polypeptide sequences provided in FIG. 3B and FIG. 13. Preferably, the region of sequence identity used for hybridization is the region of 9 nucleic acids or more encoding the region of highest conservation between the sequences shown in FIG. 13 or among eotaxins in FIG. 3B.
In another aspect, the invention features a method of producing an eotaxin polypeptide which involves: (a) providing a cell transformed with DNA encoding an eotaxin polypeptide positioned for expression in the cell (for example, present on a plasmid or inserted in the genome of the cell); (b) culturing the transformed cell under conditions for expressing the DNA; and (c) isolating the eotaxin polypeptide.
In another aspect, the invention features substantially pure eotaxin polypeptide. Preferably, the polypeptide includes a greater than 50 amino acid sequence substantially identical to a greater than 50 amino acid sequence shown in the FIG. 3A, FIG. 7, or FIG. 11, more preferably the identity is to one of the conserved regions of homology shown in FIG. 3B or FIG. 13.
In another aspect, the invention features a recombinant polypeptide capable of mediating eosinophil chemotactic events wherein the polypeptide includes a domain having a sequence which has at least 70% identity to at least one of the sequences of FIG. 3A, FIG. 7, or FIG. 12. Preferably, the identity is to the sequence in either FIG. 12. More preferably, the region of identity is 80% or greater; most preferably the region of identity is 95% or greater.
In another aspect, the invention features a method of increasing eosinophil chemotactic events wherein the method involves: (a) providing the eotaxin gene under the control of a promoter providing controllable expression of the eotaxin gene in a cell wherein the eotaxin gene is expressed in a construct capable of delivering an eotaxin protein in an amount effective to increase the eosinophil chemotactic events. The polypeptide may also be provided directly, for example, in cell culture and therapeutic uses. In preferred embodiments, eotaxin is delivered by expression of the eotaxin gene using a tissue-specific or cell type-specific promoter, or by a promoter that is activated by the introduction of an external signal or agent, such as a chemical signal or agent. In another preferred embodiment, eotaxin is delivered together with at least one other cytokine, for example, IL-4, IL-5 or both IL-4 and IL-5 together.
In preferred embodiments, the method is used for improving prognosis in patients with tumors. The method includes providing eotaxin in the region of the tumor either by providing an eosinophil attracting amount of the polypeptide or by providing an eosinophil-attracting amount of a transgene expressing the polypeptide. In one such embodiment the tumor is a solid tumor, e.g. lymphoma (e.g., Hodgkin""s), plasmacytoma, carcinoma (e.g., gastric, colonic, and lung carcinomas), melanoma, and sarcoma.
In another aspect, the invention features a method of reducing inflammation and cytotoxic damage caused by eosinophils. For example, damage occurring during asthmatic reactions, eosinophilic pneumonia, chronic obstructive pulmonary disease, bronchiectasis, cystic fibrosis, inflammatory bowel diseases (i.e., Crohn""s Disease and ulcerative colitis), eosinophilic colitis, allergic bronchiopulmonary aspergellolis, atopic dermatitis, urticaria, vasculitis (e.g., Churg-strauss disease), allergic conjunctivitis, allergic rhinitis, iatrogenic eosinophilia (e.g., responses to antibiotics and cytokine treatments such as interleukin-2 therapy), parasitic infections, idiopathic hypereosinophilic syndrome, eosinophil myalgia syndrome, eosinophil fascitis, and eosinophil cardiac disease. The method includes inhibiting eosinophil chemotaxis normally caused by eotaxin""s eosinophil attracting biological activity. Preferably, eotaxin activity is reduced using an agonist such as an anti-eotaxin antibody or eotaxin fragment. In some embodiments, the antagonist is an eotaxin polypeptide having a deletion of 1-10 N-terminal amino acids (preferably amino acids 2-8) or having an addition of 3-10 amino acids on the amino terminus (preferably 5 amino acids). Where amino acids are added they may be random or they may be selected to have particular biological properties such as stability or hydrophilicity.
In another aspect, the invention features a method of modulating histamine release via the modulation of eotaxin activity or expression. Such modulation may be accomplished using the methods described herein. For example lowering eotaxin activity may be done to decrease histamine release which occurs during anaphylaxis, urticaria, allergic conjunctivitis, allergic rhinitis.
In another aspect, the invention features a purified antibody which binds specifically to a murine or human eotaxin protein. Such an antibody may be used in any standard immunodetection method for the identification of an Eotaxin polypeptide. Such an antibody may also be used to inhibit eotaxin protein function and to predict prognosis in following tumor diagnosis. In various embodiments, the antibody may be an intact monoclonal or polyclonal antibody, but may also be an immunologically-active antibody fragment, such as an Fabxe2x80x2 or (Fabxe2x80x2)2 fragment, or a genetically engineered Fv fragment (see U.S. Pat. No. 4,946,788, hereby incorporated by reference).
In another aspect, the invention features a DNA sequence substantially identical to the DNA sequence shown in FIG. 12. In all related aspects, the invention features DNA substantially identical to the DNA sequence shown in FIG. 3A and FIG. 7.
In another aspect, the invention features a substantially pure polypeptide having a sequence substantially identical to an amino acid sequence shown in FIG. 12 and in a related aspect the invention features a substantially pure polypeptide having the sequence shown in FIG. 3A, FIG. 7, or FIG. 12.
In another aspect, the invention features an eotaxin polypeptide which has a deletion of 1-10 amino acids at the amino terminus of the mature eotaxin polypeptide (i.e., having the signed sequence cleaved). Preferably, the deletion is of amino acids 2-8 of the mature eotaxin polypeptide. In a related aspect, the invention features eotaxin polypeptides having 3-10 amino acids added to the amino terminus of the mature polypeptide. In preferred embodiments, 5 amino acids are added.
In related aspects, the invention features substantially pure nucleic acid encoding the truncated and augmented eotaxin polypeptides of the immediately foregoing paragraph.
In another aspect, the invention features a kit for detecting compounds which modulate eotaxin mediated events. The kit includes eotaxin-encoding DNA positioned for expression in a cell capable of producing a detectable eotaxin response. By eotaxin response is meant those eotaxin mediated events described herein, particularly in the examples, below.
In a related aspect, the invention features a method for detecting a compound which alters eotaxin mediated events. The method includes: i) a cell having eotaxin encoding DNA positioned for expression; ii) contacting said cell or extracts therefrom with the compound to be tested; iii) monitoring said cell or extracts therefrom for the ability to alter eotaxin mediated events, for example increased eosinophil chemotaxis.
xe2x80x9cEotaxin genexe2x80x9d means a gene encoding a polypeptide having the eotaxin eosinophil chemoattractant characteristics or other eotaxin biological activities described herein below. An eotaxin gene is a gene encoding an eotaxin polypeptide having about 60% or greater, more preferably 70% or greater amino acid sequence identity to at least one of the eotaxin polypeptide sequences of FIG. 3A, FIG. 7, or FIG. 12, or a portion thereof. For example, the gene may encode human or murine eotaxin polypeptide. An eotaxin gene may also be defined as encoding a polypeptide with at least 50% of the activity of the eotaxin polypeptides described below (preferably, such a comparison done using assay components derived from the species from which the eotaxin polypeptide to be tested is derived. Preferably, the eotaxin gene is a murine or human eotaxin gene.
xe2x80x9cEnhancing eosinophil chemotaxisxe2x80x9d means increasing the number of eosinophils in the target tissue by at least 20% relative to an untreated control tissue of similar type. Preferably, the increase in the number of eosinophils is at least two-fold.
xe2x80x9cInhibiting eosinophil chemotaxinsxe2x80x9d means decreasing the number of eosinophils in the target tissue by at least 20% relative to an untreated control tissue of similar type. Preferably, the decrease in the number of eosinophils is at least two-fold.
xe2x80x9cPolypeptidexe2x80x9d means any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
xe2x80x9cSubstantially identicalxe2x80x9d means a polypeptide or nucleic acid exhibiting at least 60%, preferably 70%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
xe2x80x9cSubstantially pure polypeptidexe2x80x9d means an eotaxin polypeptide (or other polypeptide described herein) which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight eotaxin polypeptide. A substantially pure polypeptide may be obtained, for example, by extraction from a natural source (e.g., a mammalian cell); by expression of a recombinant nucleic acid encoding an the polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., those described in column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
xe2x80x9cSubstantially pure DNAxe2x80x9d means DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
xe2x80x9cTransformed cellxe2x80x9d means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a polypeptide described herein (for example, an eotaxin polypeptide).
xe2x80x9cPositioned for expressionxe2x80x9d means that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., an eotaxin polypeptide, a recombinant protein or a RNA molecule).
xe2x80x9cPromoterxe2x80x9d means minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
xe2x80x9cOperably linkedxe2x80x9d means that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
xe2x80x9cTransgenexe2x80x9d means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
xe2x80x9cTransgenicxe2x80x9d means any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell or an animal in which a gene has been inactivated by artifice. As used herein, the transgenic organisms are generally transgenic rodents and the DNA (transgene) is inserted by artifice into the nuclear genome or in which a gene has been inactivated.
xe2x80x9cConserved regionxe2x80x9d means any stretch of six or more contiguous amino acids exhibiting at least 30%, preferably 50%, and most preferably 70% amino acid sequence identity between two or more of the eotaxin family members.
xe2x80x9cDetectably-labelledxe2x80x9d means any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labelling a molecule are well known in the art and include, without limitation, radioactive labelling (e.g., with an isotope such as 32p or 35S) and nonradioactive labelling (e.g., chemiluminescent labelling, e.g., fluorescein labelling).
xe2x80x9cTransformationxe2x80x9d means any method for introducing foreign molecules into a cell. For example, molecules may be introduced using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., chloroplasts and mitochondria), bacteria, yeast, fungi, algae, and animal tissue.
xe2x80x9cPurified antibodyxe2x80x9d means antibody which is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., an eotaxin-specific antibody. A purified eotaxin antibody may be obtained, for example, by affinity chromatography using recombinantly-produced eotaxin protein or conserved motif peptides and standard techniques.
xe2x80x9cModulatory compoundxe2x80x9d, as used herein, means any compound capable of either increasing eotaxin expression (i.e., at the level of transcription, translation, or post-translation) or increasing eotaxin protein activity (i.e., the amount of activity, for example, eosinophil chemotaxis, per unit of eotaxin protein).
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawings will first be described.